Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, β-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model.

The basicity of highly protonated cytochrome c (cyt c) and myoglobin (myo) ions were investigated using tandem mass spectrometry,i on-molecule reactions (IMRs), and theoretical calculations as af unction of charge state. Surprisingly,h ighly charged protein ions (HCPI) can readily protonate non-polar molecules and inert gases,i ncluding Ar, O 2 ,a nd N 2 in thermal IMRs.T he most HCPIs that can be observed are over 130 kJ mol À1 less basic than the least basic neutral organic molecules known( tetrafluoromethane and methane). Based on theoretical calculations,itispredicted that protonated cyt ca nd myo ions should spontaneously lose ap roton to vacuum for charge states in which every third residue is protonated. In this study,H CPIs are formed where every fourth residue on average is protonated. These results indicate that protein ions in higher charge states can be formed using al ow-pressure ion source to reduce proton-transfer reactions between protein ions and gases from the atmosphere.Electrospray ionization (ESI) is renowned for its ability to form intact, gaseous,m ultiply charged protein ions for rapid and sensitive detection by mass spectrometry.[1] However,the mechanism by which protein ions are formed in ESI is controversial and continues to be actively debated. Thet wo primary competing models to explain ion formation are known as the charge residue model (CRM) [2] and the ion evaporation model (IEM).[3] In both models,a sn eutral molecules evaporate from ac harged droplet, the electric field at the surface of the droplet increases,w hich initiates fission.[4] Such droplet fission events result in the emission of afine stream of smaller droplets that remove less than 1% of the mass but more than 30 %o ft he charge of the precursor droplet. [4,5] In the CRM, sequential droplet evaporation and Coulombic fission events yield acharged droplet that contains asingle analyte ion, which evaporates to dryness via the loss of neutral solvent molecules.Inthe IEM, the electric field on the surface of ah ighly charged droplet near the moment of ion formation is sufficient to result in the ejection of an analyte ion from the surface of the ionic droplet. Themajority of current evidence indicates that fully desolvated protein ions formed from buffered aqueous solutions are formed by the CRM. Charge carriers such as solvated hydronium ions can be lost via ion evaporation during the ESI process. [6] Recently,the chain-ejection model (CEM), [7] which is related to the IEM, was proposed to explain the formation of protein ions from denaturing solutions based on results from molecular dynamics simulations. [7] In the CEM, ad enatured, disordered protein chain is ejected from ah ighly charged, nanometer-sized ionic droplet. As the protein ion protrudes and is ejected from the droplet, proton transfer to the protein ion can occur. However,t he mechanism by which highly charged protein ions (HCPIs) are formed from denaturing solutions is less well established with evidence supporting both the CRM [8] and CEM/IEM h...

The effects of 12 acids, 4 solvents, and 8 low-volatility additives that increase analyte charging (i.e., superchargers) on the charge state distributions (CSDs) of protein ions in ESI-MS were investigated. We discovered that (i) relatively low concentrations [5% (v/v)] of 1,2-butylene carbonate (and 4-vinyl-1,3-dioxolan-2-one) can be added to ESI solutions to form higher charge states of cytochrome c and myoglobin ions than by using more traditional additives (e.g., propylene carbonate, sulfolane, or m-nitrobenzyl alcohol) under these conditions and (ii) the width of CSDs narrow as the effectiveness of superchargers increase, which concentrates protein ions into fewer detection channels. The use of strong acids (pKa values < 0) results in essentially no protein supercharging, higher adduction of acid molecules, and wider CSDs for many superchargers and proteins, whereas the use of weak acids (pKa > 0) results in significantly higher protein ion charging, less acid adduction, and narrower CSDs, indicating that protein ion supercharging in ESI can be significantly limited by the binding of conjugate base anions of acids that neutralize charge sites and broaden CSDs. The extent of protein charging as a function of acid identity (HA) does not strongly correlate with gas-phase proton transfer data (i.e., gas-phase basicity and proton affinity values for HA and A(-)), solution-phase protein secondary structures (as determined by circular dichroism spectroscopy), and/or acid molecule volatility data. For protein-denaturing solutions, these data were used to infer that the "effective" pH of ESI generated droplets near the moment of ion formation can be ∼0, which is ca. 1 to 3 pH units lower than the pH of the solutions prior to ESI. Electron capture dissociation (ECD) of [ubiquitin, 17H](17+) resulted in the identification of 223 cleavages, 74 of 75 inter-residue sites, and 92% ECD fragmentation efficiency, which correspond to highest of these values that have been obtained by ECD of a single isolated charge state of ubiquitin.

Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complimentary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum.

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed.Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that do not contain the C-terminus or N-terminus of the protein. Conventionally, internal fragments have been disregarded as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50% to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments. File list (2)download file view on ChemRxiv Internal Fragment Manuscript_II.pdf (464.44 KiB) download file view on ChemRxiv Supporting Information for Internal Fragment Manuscript...

No abstract

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N-or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photonbased fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15−20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

No abstract